Due to the unacceptable toxicity and ineffectiveness of currently available cancer chemotherapy and radiation, the search for new molecular targets is in high gear. While initially identified as a promoter of the autophagy pathway, UVRAG (UV irradiation-resistance associated gene) has now emerged in our recent studies as a bona fide guardian of the genome. Specifically, we have discovered that this protein mediates the repair of damaged DNA by targeting DNA-PK and patrols centrosome stability by targeting CEP63 in a manner independent of its role in autophagy signaling. We propose that UVRAG is a novel regulator of chromosomal stability, supporting a direct causal role for UVRAG inactivation in cancer development. To the best of our knowledge, these studies represent the first description of an autophagy-related factor directly functioning in centrosome regulation and DNA repair to maintain genome integrity. Thus, the proposed research will expand on this knowledge to comprehensively assess the molecular mechanisms by which UVRAG interacts with DNA-PK to activate DNA repair upon double-strand breaks (DSBs) (Aim 1) and targets CEP63 to prevent centrosome amplification (Aim 2), thereby maintaining both structural and numeric chromosomal integrity of cells. Furthermore, to determine the in vivo contribution and specific roles of chromosomal instability and autophagy in UVRAG-associated tumor suppression (Aim 3), we will develop a drug-inducible transgenic murine model, which faithfully recapitulates an oncogenic lesion of UVRAG found in human cancers. These aims will be addressed using multidisciplinary innovative approaches that integrate state-of-the art genetic, biochemistry, quantitative live-cell imaging, and physiological assays in cells and in mice with targeted mutations in genes that are related to UVRAG and required for the regulation of genomic stability and autophagy. Results gained from this study will undoubtedly illuminate new views on UVRAG as an autophagic tumor suppressor and a protector of the genome, reveal functional interactions between UVRAG, autophagy, and genomic instability, and also hold promise for the development of novel therapeutics for cancer.